Recruitment, adhesion, and aggregation of platelets at sites of vascular injury are critical to generation of beneficial blood clotting events. However, excessive accumulation of platelets, e.g., at sites of ruptured atheriosclerotic plaques, can give rise to the development of acute coronary syndromes, stroke, ischaemic complications of peripheral vascular disease, and other disease states. Fuster, V., Badimon, L., Badimon, J. J. & Chesebro, J. H. The Pathogenesis of Coronary Artery Disease and the Acute Coronary Syndromes (1). N. Engl. J. Med. 326, 242-250 (1992); Falk, E, Shah, P. K. & Fuster, V Coronary Plaque Disruption. Circulation 92, 657-671 (1995). Promise for enhanced clinical management of such vascular diseases has arisen in recent years with progress in understanding of the mechanisms underlying the formation of arterial plaque and thrombosis and of the criticality of the role of platelet activity in the development of cardiovascular disease.
Tempered by the understanding that antithrombotic treatment should be effective and yet avoid undermining hemostasis, clinicians of cardiovascular disease prevention and treatment have depended on mild therapeutic agents like aspirin and clopidogrel for widespread application. There are a variety of other antithrombotic drugs, including coumadin and abciximab (ReoPro®), ticlopidine, and others, but there remains an urgent need for newer and safer antithrombotics, to address stroke, deep vein thrombosis (DVT), myocardial infarction, coronary artery disease, cerebrovascular disease, peripheral arterial disease, diabetes mellitus, atrial fibrillation, congestive heart failure, and other vascular disorders. Jackson S P and Schoenwaelder S M. Antiplatelet Therapy: In Search of the ‘Magic Bullet’. Nat. Rev. Drug. Discov. 2 (10), 775-89 (2003). Review. More versatile and effective and yet selective and safe therapeutic agents are currently the object of extensive research worldwide, especially in light of the increasing prevalence of cardiovascular disease both due to changes in diet and lifestyle and in view of the aging of the population. Special emphasis has been placed on the issue of improving efficacy without compromising safety, since all forms of presently available antithrombotic therapies cannot be administered at potent doses without producing negative physiological conditions, primarily bleeding events.
Upon vessel injury and attendant removal or damage of the protective endothelial lining, platelets encounter a diverse set of proteins from the connective tissue of the vessel wall. These include collagen and von Willebrand factor (vWf). Platelet adhesion to these proteins and subsequent activation is mediated by a multitude of platelet receptors. Adhesion of platelets to the extracellular matrix triggers a series of signaling events that ultimately result in formation of a hemostatic plug known as a thrombus. Recent findings provide strong evidence that immediately following vessel rupture, the platelet receptor GPVI binds loosely to exposed collagen, which is alone insufficient to induce stable platelet adhesion, but which triggers a tyrosine kinase-based signaling pathway that results in major conformational changes and attendant activation in specific receptors, including integrin α2β1. Emsley J, Knight C G, Farndale R W, Barnes M J, Liddington R C. Structural Basis of Collagen Recognition by Integrin Alpha2Beta1. Cell. 101 (1), 47-56 (2000).
Integrin α2β1, also known as platelet GPIa-IIa, was the first collagen receptor to be identified on platelets. Nieuwenhuis H K, Akkerman J W, Houdijk W P, Sixma J J. Human Blood Platelets Showing No Response to Collagen Fail to Express Surface Glycoprotein Ia. Nature. 318 (6045), 470-2 (1985); Santoro S A. Identification of a 160,000 Dalton Platelet Membrane Protein That Mediates the Initial Divalent Cation-dependent Adhesion of Platelets to Collagen. Cell. 46 (6), 913-20 (1986). Similar to other members of the integrin family, α2β1 inks the cytoskeleton of the cell with the extracellular matrix. Hynes R O. Integrins: bidirectional, allosteric signaling machines. Cell. 110 (6):673-87. Review (2002). Besides playing an essential role in adhesion to the extracellular matrix, integrins are indispensable for cellular signaling. All integrins are heterodimers, consisting of an α subunit and a β subunit. About half of the known mammalian integrins, including α2β1, have an I-domain inserted into the α subunit (Hynes, 2002). In these cases, the I-domain is responsible for binding of the integrin to its natural ligand(s). A specific amino acid sequence in collagen, GFOGER (O=hydroxyproline), promotes stable binding to the I-domain of α2β1. Onley D J, Knight C G, Tuckwell D S, Barnes M J, Farndale R W. Micromolar Ca2+ concentrations are essential for Mg2+-dependent binding of collagen by the integrin alpha 2beta 1 in human platelets. J Biol. Chem. 275 (32):24560-4 (2000). Binding occurs in a cation dependent manner, supported by either magnesium or manganese Tuckwell D, Calderwood D A, Green L J, Humphries M J. Integrin alpha 2 I-domain is a binding site for collagens. J Cell Sci. 108 (Pt 4):1629-37 (1995). A crystal structure of a complex between the I-domain of α2β1 and a triple helical peptide containing the GFOGER sequence has been solved. Emsley J, Knight C G, Farndale R W, Barnes M J, Liddington R C. Structural basis of collagen recognition by integrin alpha2beta1. Cell. 101 (1), 47-56 (2000). A glutamic acid (E) from the middle strand of the triple helix coordinates to metal-ion dependent adhesion site (MIDAS) while other residues of the GFOGER motif from the middle and trailing strands interact with complementary sites on the I-domain surface.
Importantly, integrin α2β1 has multiple states of activation which can be regulated from inside or outside of the cell. Hynes R O. Integrins: bidirectional, allosteric signaling machines. Cell. 110 (6):673-87. Review (2002). For instance, signaling through the platelet receptor GPVI impinges upon the cytoplasmic domain of α2β1, which results in a dramatic conformational change that eventually propagates along the α2β1 integrin, ultimately affecting the I-domain at the integrin's head. Integrin activation is induced by several other platelet agonists, including ADP and thrombin. Jung S M, Moroi M. Platelets interact with soluble and insoluble collagens through characteristically different reactions. J. Biol Chem. 273 (24):14827-37 (1998). The activated integrin can than bind tightly to collagen. This adhesion can potentially be blocked with either a direct competitor of the collagen/I-domain interaction or with an allosteric regulator, the latter of which precludes activation of the I domain. Two types of small-molecule inhibitors have been developed for a related integrin, αLβ2. Shimaoka M, Salas A, Yang W Weitz-Schmidt G, Springer T A. Small molecule integrin antagonists that bind to the beta2 subunit I-like domain and activate signals in one direction and block them in the other. Immunity. 19 (3):391-402 (2002). The first binds to the I-domain of αLβ2 at a distant site from the MIDAS, blocking activation of its I domain and subsequent binding to ICAM-1. The second binds to the I-like domain of the β subunit, which is located directly beneath the I domain. A direct competitive inhibitor of an I-domain/ligand interaction has not yet been reported.
Despite the fact that α2β1 integrin was discovered more than 15 years ago, its precise role in platelet adhesion and aggregation remains controversial. This is partially due to the overlapping functions of α2β1 and GPVI. Chen H, Kahn M L. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. Mol Cell Biol. 23 (14):4764-77 (2003). Integrin α2β1 is essential for platelet adhesion and activation on monomeric type I collagen; it has been demonstrated through platelet analysis that adhesion and thrombus growth on pepsin-solubilized type I collagen under low and high shear flow conditions is absolutely dependent on functional α2β1. Savage B, Ginsberg M H, Ruggeri Z M. Influence of Fibrillar Collagen Structure on the Mechanisms of Platelet Thrombus Formation Under Flow. Blood. 94 (8), 2704-15 (1999); Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mokhtari-Nejad R, Lindhout T, Heemskerk J W, Zirngibl H, Fassler R. Glycoprotein VI But Not Alpha2Beta1 Integrin is Essential For Platelet Interaction With Collagen. EMBO J. 20 (9), 2120-30 (2001). However, on the more physiologically relevant insoluble collagen (fibrillar collagen), α2β1 integrin may be dispensable, at least in the context of hemostasis. Nieswandt B, Watson S P. Platelet-Collagen Interaction: Is GPVI the Central Receptor? Blood. 102 (2), 449-6 (2003). Review. For instance, fibrillar collagen-induced aggregation of β1-null mouse platelets is not reduced, despite a slight time delay. Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mokhtari-Nejad R, Lindhout T, Heemskerk J W, Zirngibl H, Fassler R. Glycoprotein VI But Not Alpha2Beta1 Integrin is Essential For Platelet Interaction With Collagen. EMBO J. 20 (9), 2120-30 (2001). Furthermore, the β1-null platelets adhere normally to fibrillar collagen under static conditions. Nonetheless, it has been established that adhesion under physiological conditions of blood flow requires a functional α2β1 integrin. Siljander P R, Munnix I C, Smethurst P A, Deckmyn H, Lindhout T, Ouwehand W H, Farndale R W, Heemskerk J W Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood. Blood. 103 (4):1333-41 (2004).
Studies of platelets derived from two individuals with an integrin α2β1 deficiency have demonstrated a defect in adhesion and spreading on the subendothelium. Nieswandt B, et al. (2001); Ruggeri Z M. Platelets In Atherothrombosis. Nat. Med. 8 (11), 1227-34 (2002). Review. Indeed, these patients exhibit only modest degree of defect in hemostasis, manifested as only minor bleeding complications. Nieuwenhuis H K, et al., Nature. 318 (6045), 470-2 (1985); Nieuwenhuis H K, Sakariassen K S, Houdijk W P, Nievelstein P F, Sixma J J. Deficiency of Platelet Membrane Glycoprotein Ia Associated With a Decreased Platelet Adhesion to Subendothelium: A Defect in Platelet Spreading. Blood. 68 (3), 692-5 (1986). This has important implications for the search for antithrombotic therapies with favorable safety profiles. It suggests that antagonism of α2β1 integrin will have a beneficially mild antithrombotic effect; increasing amount of evidence indeed suggests that α2β1 may have a greater role in pathological thrombosis relative to normal hemostasis. This observation may reflect the fact that an increased amount of collagen accumulates in diseased blood vessels. For instance, the extracellular matrix around an atheroslerotic lesion is heavily enriched in collagens. Nieswandt B, et al., (2003). Besides providing an adhesive support for platelets, collagen sends potent prothrombotic signals into the cell through interaction with its platelet receptors. Overexpression of α2β1 integrin has been linked to cardiovascular disease in humans. Kritzik M, Savage B, Nugent D J, Santoso S, Ruggeri Z M, Kunicki T J Nucleotide polymorphisms in the alpha2 gene define multiple alleles that are associated with differences in platelet alpha2 beta1 density. Blood. 92 (7):2382-8 (1998). Furthermore, recent in vivo data indicates that α2β1-deficient mice have delayed thrombus formation following carotid artery injury. He L, Pappan L K, Grenache D G, Li Z Tollefsen D M, Santoro S A, Zutter M M. The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo. Blood. 102 (10):3652-7 (2003). These data reveal a critical role for α2β1 in thrombosis. Hence, the α2β1 integrin is an important pharmacological target for cardiovascular diseases, and the resulting treatment is expected to be well-tolerated and provide long-term antithrombotic protection.
Equally significant, the α2β1 integrin may be a target for cancer, several types of viral infections, and other pathologies. Overexpression of α2β1 in various types of cancer cells, particularly in human melanoma cells and hepatocellular carcinomas, has been linked to tumor metastasis. Han J. Jenq W, Kefalides N A. Integrin Alpha2Beta1 Recognizes Laminin-2 and Induces C-erb B2 Tyrosine Phosphorylation in Metastatic Human Melanoma Cells. Connect Tissue Res. 40 (4), 283-93 (1999). Yang C, Zeisberg M, Lively J C, Nyberg P, Afdhal N, Kalluri R. Integrin Alpha1Beta1 and Alpha2Beta1 Are the Key Regulators of Hepatocarcinoma Cell Invasion Across the Fibrotic Matrix Microenvironment. Cancer Res. 63 (23), 8312-7 (2003). The α2β1 integrin is known to be the primary melanoma cell adhesion molecule for type IV collagen, indicating a key role for that integrin in pathological metastasis Knutson J R, Iida J, Fields G B, McCarthy J B. CD44/Chondroitin Sulfate Proteoglycan and Alpha 2 Beta 1 Integrin Mediate Human Melanoma Cell Migration on Type IV Collagen and Invasion of Basement Membranes. Mol Biol Cell. 7 (3), 383-96 (1996). Ligand binding by the α2β1 integrin triggers a series of intracellular signaling events that ultimately result in the release of cytokines and proteases, both of which are beneficial for tumor cell progression. Baronas-Lowell D, Lauer-Fields J L, Borgia J A, Sferrazza G F, Al-Ghoul M, Minond D, Fields G B. Differential Modulation of Human Melanoma Cell Metalloproteinase Expression by Alpha2Beta1 Integrin and CD44 Triple-Helical Ligands Derived from Type IV Collagen. J Biol Chem. 279 (42), 43503-13 (2004). Furthermore, antagonism of the α2β1 integrin suppresses angiogenesis. Senger D R, Perruzzi C A, Streit M, Koteliansky V E, de Fougerolles A R, Detmar M. The Alpha(1)Beta(1) and Alpha(2)Beta(1) Integrins Provide Critical Support For Vascular Endothelial Growth Factor Signaling, Endothelial Cell Migration, and Tumor Angiogenesis. Am J Pathol. 160 (1), 195-204 (2002). This has profound implications since angiogenesis is involved in growth and metastasis of solid tumors, rheumatoid arthritis, diabetic retinopathy, and a variety of other important disease states. Folkman J. Angiogenesis in Cancer, Vascular, Rheumatoid and Other Disease. Nat. Med. 1 (1), 27-31 (1995). Review; Senger D R, Van de Water L, Brown L F, Nagy J A, Yeo K T, Yeo T K, Berse B, Jackman R W, Dvorak A M, Dvorak H F. Vascular Permeability Factor (VPF, VEGF) in Tumor Biology. Cancer Metastasis Rev. 12 (3-4), 303-24 (1993). Review; Ferarra, N. The Role of Vascular Endothelial Growth Factor in Pathological Angiogenesis. Breast Cancer Res Treat. 36 (2), 127-37 (1995). Review. Specific blocking of α2β1 function halts capillary morphogenesis, the essential antecedent to angiogenesis, whereas blocking of related integrin dimers or monomer subunits does not similarly arrest morphogenesis. Sweeney S M, DiLullo G, Slater S J, Martinez J, Iozzo R V, Lauer-Fields J L, Fields G B, San Antonio J D. Angiogenesis in Collagen I Requires Alpha2Beta1 Ligation of a GFP*GER Sequence and Possibly p38 MAPK Activation and Focal Adhesion Disassembly. J Biol Chem. 278 (33), 30516-24 (2003). Antagonism of the α2β1 integrin also curbs haptotactic endothelial cell migration, Senger D R et al., a critical step in extravasation of tumor cells into secondary tissues.
It has also recently been shown that human cytomegalovirus (HCMV), which is extremely promiscuous and responsible for significant mortality, requires the presence of α2β1 to penetrate a cell. Feire A L, Koss H, Compton T. Cellular Integrins Function as Entry Receptors For Human Cytomegalovirus Via a Highly Conserved Disintegrin-Like Domain. Proc Natl Acad Sci USA. 101 (43), 15470-5 (2004). Likewise, integrin α2β1 has been strongly implicated in rotavirus cell attachment and entry. Graham K L, Halasz P, Tan Y, Hewish M J, Takada Y, Mackow E R, Robinson M K, Coulson B S. Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognize alphaXbeta2 and alphaVbeta3 by using VP7 during cell entry. J Virol. 77 (18), 9969-78. (2003). Rotaviruses are leading causes of acute gastroenteritis in human infants and young children and animals around the globe. Id. It has been demonstrated that inhibition of the α2β1 integrin forestalls cell binding and infection by rotaviruses. Londrigan S L, Graham K L, Takada Y, Halasz P, Coulson B S. Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit. J Virol. 77 (17), 9486-501 (2003). Similarly, viruses of the Piconaviridae family, such as Echovirus 1 (Echo 1), have also been shown to utilize the α2β1 integrin during the cell-infection cycle. Triantafilou K & Triantafilou M. A biochemical approach reveals cell-surface molecules utilised by Picornaviridae: Human Parechovirus 1 and Echovirus 1. J Cell Biochem. 80 (3), 373-81 (2001). Echo viruses are implicated in numerous human pathologies; for example, certain forms of aseptic meningitis and acute respiratory illness are known to be caused by the Echo-1 virus. See, e.g., Kumar R. Aseptic meningitis: Diagnosis and management. Indian J Pediatr. 72 (1), 57-63 (2005).
Inhibition of the α2β1 integrin may prove effective in impeding binding and entry of these problematic and medically-significant viruses, and in treatment of cancers and other disease states concerning which α2β1 expression and functionality is a significant factor, and previous efforts have been made to provide compounds possessing α2β1 integrin inhibitory activity. See Takayanagi, M et al., WO 03/008380. As yet, however, there is an unfulfilled need in these respects. The present invention provides inhibitors of integrin α2β1 and methods for their synthesis and use.